Abstract
SAK-HV is an anti-atherosclerosis recombinant fusion protein developed by our lab. Our study determined that SAK-HV promoted macrophage proliferation, of which the mechanism was explored by both RAW264.7 cells and primary macrophages. Mass spectrometric analysis and co-immunoprecipitation were combined to screen the SAK-HV-interacting proteins in RAW264.7 cells. Confocal microscopy was adopted to detect the localization of SAK-HV in cells. The results indicated that SAK-HV triggered macrophage proliferation via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) pathways by its SAK-mutant functional domain. We screened out Uba1 as the SAK-HV-interacting protein in the RAW264.7 cells and discovered their co-localization in the cytoplasm and nucleus. Inhibiting Uba1 significantly decreased the SAK-HV-induced macrophage proliferation. Thus, we postulated an attractive model of ubiquitination, in which the interactions between Uba1 and specific E2 enzymes are blocked by its interaction with SAK-HV. Based on this model, we detected the decreased self-ubiquitination of MEKK1 after SAK-HV treatment and concluded that SAK-HV inhibits the self-ubiquitination of MEKK1 via its SAK-mutant functional domain to activate MAPK/ERK and JNK pathways, promoting macrophage proliferation. This conclusion highly supported our hypothesized model of ubiquitination at the level of Uba1, which may represent a novel paradigm to promote macrophage proliferation by using the E1 enzyme (Uba1) as a switch.
Highlights
Macrophages are found in all tissues and play important roles in development, homeostasis, tissue repair, and immunity [1]
Macrophage proliferation is involved in many bbiiological processes and tthhe mmeecchhaanisms oof initiating proliferation have become a focus in rreecceennt yyeeaarrss [[3300––3322]]
Our study illustrated the mechanism of SAK-HV-promoted macrophage pprroolliiffeerraattiioonn, iinn wwhhiicchh UUbbaa11 mmiigghhtt ppllaayy aanniimmppoorrttaannttrroollee
Summary
Macrophages are found in all tissues and play important roles in development, homeostasis, tissue repair, and immunity [1]. RmAaWcro2p64h.a7gmesu, rminoeusmeaecmrobprhyaogneisc, limveorusceellemlinbery(BonNicL-lCivLe2r),caenlldlihnuem(BaNn La-cCuLte2)m, oanndocyhtuicmlaenukaecmutiea mceollnolicnyeti(cTHleuPk-1e)m, tiao csecrlleelinnefo(rTpHoPte-1n)t,iatol tsacrrgeeetncefollrlipnoetse.ntUianl etxaprgeecttecdellyl, lwineesf.oUunndextpheactteSdAlyK,-HwVe fsotiumnudlattihoant coSuAlKd-sHelVectisvtiemlyuilnadtiuocne pcrooulilfderasteilveecteifvfeeclyts iinndRuAcWe 2p64ro.7limfearacrtoivpehaegfefse,cTtsHPin-1 RmAonWo2c6y4te.7s, manadcrporpihmaagreys,pTeHritPo-n1emalomnoaccryotepsh,aagnedceplrlism; haorywpeveerirt,otnheisalacmtiavcirtoypwhaags enocet lolsb;sherovweedvienr,ththeisotahcetrivcietyll lines We used both the RAW264.7 cell line and primary macrophages to investigate the mechanism of macrophage proliferation promoted by SAK-HV. 5D).uDriungrinthgetshceresecnreinegn,inwge, sweelecsteeledcfteodr ifnohribinithoirbcitoonrcecnontrcaetniotrnastitohnast sthhoatwsehdotwheedcltohseesctlionshesibt itinorhyibeiftfoercytsetfofetchtse ctoontthreolcgornoturopl, wgrhoiulep,awvohiidleinagveoxidciensgsivexecienshsiivbeitiionnhiibnitoiordneirntoorcdheerctko tchheecaknttahgeoannistamgobneitswmeebnetSwAeKen-HSVAKa-nHdVthaensde itnhhesibeitinorhsi.biTtohres.sTehleectseedleicntehdibiintohribciotonrcecnotnrcaetinotnrastiwonersewuesreedutsoedinvtoesitnigvaetsetigthaeteetfhfeectesffoecftEs RoKf EaRnKd JaNndKJaNcKtivaacttiiovnatioonnSoAnKS-AHKV-H-inVd-uincdeudcpedroplirfoelriafetiroanti.onT.hTehreerseuslutsltsshsohwowededththatatininhhibibitiotorrssooffbbootthh tthhee EERRKK aanndd JJNNKK ppaatthhwwaayyss ccoouulldd ssiiggnniiffiiccaannttllyy iinnhhiibbiitt SSAAKK--HHVV--iinndduucceedd pprroolliiffeerraattiioonn iinn RRAAWW226644..77 cceellllss ((FFiigguurree 55)). TThhiiss ffuurrtthheerr vveerriiffiieedd tthhee kkeeyy rroolleess ooff tthhee EERRKK aanndd JJNNKK ppaatthhwwaayyss iinn tthhee SSAAKK--HHVV--iinndduucceeddpprroolilfieferaratitoionnofoRf RAAWW26246.74.c7ecllesl.lsB.eBsiedseids,ews,ewaelsoalosobsoebrvseerdvtehde tahcetivaacttiivonatoiofnERofKEaRnKd and JNK pathways in primary peritoneal macrophage cells from C57BL/6J mice after SAK-HV. TThhee SSAAKK--HHVV--eennhhaanncceedd pprroolliiffeerraattiioonn eeffffeeccttss iinn bbootthh tthhee RRAAWW226644..77 aanndd pprriimmaarryy mmaaccrroopphhaaggee cceellllss wweerree rreemmaarrkkaabbllyy aammeelliioorraatteedd bbyy PPAARR--4411 ((FFiigguurree 1100BB–,CC)). RRaaww226644..77 cceellllss wweerree ttrreeaatteedd wwiitthh SSAAKK--HHVV ffoorr00..55hhaattmmuullttiipplleeccoonncceennttrraattiioonnss((00..11,,11,,aanndd55μμMM))sseeppaarraattiivveellyyininppaanneellCC
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
